Centromeres Flashcards
What is the main function of centromeres and what are the consequences of a chromsome having more or less than one?
Essential for the normal segregation of chromosomes. Centromeres must attach equally to microtubules to be pulled apart equally.
Acentric chromsomes are lost becasue they segregate randomly
Polycentric chromsomes result in anaphase bridge formation and breakage.
What are the structures of centromeres?
171bp tandem repeat arrays of alpha satellites. All the DNA is in condensed heterochromatin, this can be seen with C banding during metaphase.
The centromere contains a kinetochore which is a protein complex associated with the centromere during mitosis and is where the microtubules attach and pull apart sister chromatids.
Describe alpha satellites
They are AT richcontaining many CpG islands for methylation. Alpha satellites can contain repeats within repeats temred HORs - Higher order repeat structures).
Each side of the alpha satellites are flanked by other alpha satellite repeats some of which are reversed. Some of these other flanking repeats contain CENP-B box motifs which binds CENP-B.
Where do centromeres form?
Centromeres from in some but not all HORs. The location within the HORs can vary but once formed it is conserved between cell divisions.
What are pericentric satellites?
These are flanking structures formed from other repeats, they are polymorphic and vary between centromeres and species.
Both pericentric and centric satellites recruit cohesin molecules to hold the sister chromatids together until anaphase - these become especially important during meiosis.
Is centromere formation reliant on the DNA sequence?
NO
Sequence is not conserved between species
Dicentric chromosomes have only 1 active centromere
2 strands of same DNA do not have same centromere competance.
Neo centromeres can form on non-centromeric DNA - example being in cancer and on chromosome 10 where deletion of centromeric DNA has occured and new centromere formed on normal DNA
However throughout species the DNA chosen is usually AT rich Formation is dependant on epigenetic markers that establish centromere heterochromatin.
What epigenetic markers are important for centromere formation?
Histone tails protrude from histone structure and can be modified. Tri methylation of lysine 9 on histone 3 causes heterochromatin protein 1 to binds (HP1 - non histone structural component of heterochromatin)
Methylation of lysine 27 on histone 3 results in transcriptional silencing and is found on pericentric regions.
Centromere core distinct from pericentromere - histone sometimes replaced with CENP-A which cannot be modified. When packaged CENP-A is on the outside facing direction of microtubules. Also H3 has methylation of lysine 4 and 36 indicating they are transcriptionally active.
What does CENP-C do?
CENP-C binds to CENP-A and can also binds DAN directly. It is specific to active centromeres. Both are essential for proper mitotic segregation and cell survival.
What are the kinetochore proteins
There are 100s of proteins involved in the kinetochore
The main inner centromere proteins are: CENP-A and H3 are the important ones for binding DNA. CPC Chromosome passenger complex is involved in binding sister chromatids together.
CENP-B binds to the motifs on the alpha satellites. Probably involved in stabilising kinetochore and recruiting CENP-C as when knocked out there is no change in mice and the Y chromosomes no domains.
CCAN - constituitive centromere associated network - 16 proteins - most are essential and present throughout out the cell cycle.
Outer kinetochore proteins - these are only recruited to CCAN durng mitosis. They are required to bind to micro tubules
How is the centromeric position maintained?
In S.Pombe is was noted that the outer repeats of centromeres are transcriptionally active. The RNA produced is not translated but enters the interference pathway forming siRNAs. They associate with RITs complex (RNA induced initiation of transcriptional gene silencing).
These siRNAs find new transcripts coming from the centromeres and recruits histone modification enzymes which put heterochromatic marks onto the centromere DNA
Is transcriptional activity also seen in vertebrates?
Yes - RNA Polymerase II seen associated with the active kinetochore. Long non coding strands from the pericentric repeats recruit methylation proteins to maintain the pericentric heterochromatin.
Are the alpha satellites transcriptional active?
Yes transcripts also seen coming off these structures.
This has two function:
- As the polymerase goes through this helps to remove H3 and deposit new CENP-A.
- The RNA produced forms R loops which recruit RPA and regulates an ATR DNA damage response allowing the cell to enter anaphase.
When does CENP-A loading occur?
CENP-A loading only occurs once per cell cycle. Every replication causes it to dilute. The time that CENP-A is loaded is not conserved between species.
Humans load early in G1 and requires a chaperone. This is odd as it means loading takes place before DNA replication meaning it goes through mitosis with half the normal amount of CENP-A.
How is disjunction prevented
Cohesins bind sister chromatids together - to be seperated these must be digested or risk non disjunction.
Rad21 (part of cohesin complex) is cleaved by seperase. Seperase is normally inhibted by securin.
If a kinetochore is not bound to microtubules then there is rapid recruitment of Mad2. Mad2 binds to CDC20 (anaphase promoting complex). This would normally ubiquinate securin targeting it for destruction.When microtubules does binds Mad2 stops being released.
